Since immunoassay was first described by Berson and Yalow in the late 1960's, a number of immunoassays with different assay formats (e.g. competitive vs. noncompetitive), different separation techniques (e.g., secondary antibody separation vs. solid phase separation), and different labels (e.g., isotopic vs. non-isotopic), have been developed for the detection of a wide spectrum of substances including molecules as small as digoxin and as large as a virus. Until recently, all these assays employed only one signal-generating label at a time which resulted in the limitation of detecting one substance (analyte) in one measurement.
The use of two radioisotopes, i.e., iodine 125 and cobalt 57, in a radioimmunoassay for detecting two analytes simultaneously in a single measurement has been demonstrated in a Vitamin B 12/Folate test (U.S. Pat. No. 4,146,602, Gutcho et al.) a TSH/ Free T4 test (Simultrac.TM. FT4/TSH, Becton Dickinson Immunodiagnostics), and the LH/FSH test (ComboStat.TM. LH/FSH, Micromedic Systems, ICN). In these assays, the discrimination between radioisotopes in the measurement is based on differences in their energy spectra.
The use of two fluorophores, e.g., fluorescein and rhodamine, in a qualitative immunoassay for detecting two substances simultaneously in a single sample but not in a single measurement has been demonstrated in the cell sorting techniques (FACS.TM. Cell Sorting System, Becton Dickinson). Recently, the use of two fluorophores, i.e. fluorescein and phycoerythrin, in a quantitative immunoassay for detecting human IgG and IgM in a single sample but not in a single measurement, was reported (R. Houghton, Abstract, Clin. Chem. Vol. 32, p. 1067, 0 1986). In these fluorescence-based immunoassays, the resolution of the fluorophores in the measurement is based on the differences in their excitation and emission spectra.
A method of time-resolved immunoassay is described in U.S. patents 4,058,732 and 4,374,120. In this method a fluorescent probe is employed that has a fluorescence decay (lifetime) that substantially exceeds the duration of the exciting pulse and the duration of the background non-specific fluorescence. A time-gating is used to reduce the background fluorescence, i.e., the measurement of the fluorescence is delayed until a certain time has elapsed from the moment of excitation. The delay time is sufficiently long for the background fluorescence to have ceased. When the fluorescence signal is measured (after the delay) the measurement is an integrated measurement, i.e. all the light arriving at the detector during the measuring period is measured without regard to the time of arrival. The purpose of this delayed measurement is to ensure that only one fluorescence signal reaches the detector during measurement.